Rhesus CD34 Research Articles

TRIM5α Variation Affects Lentiviral Transduction Efficiency for Long-Term Hematopoietic Repopulating Cells in a Rhesus Stem Cell Transplantation Model

Abstract 2056The Tripartite Motif-containing protein 5 α (TRIM5α) is thought to play an important role in restricting retroviral infection among species in nature, and restricts HIV-1 based lentiviral transduction of rhesus hematopoietic repopulating cells. We previously developed a chimeric HIV-1 based lentiviral vectors (χHIV vectors) which includes a simian immunodeficiency virus (SIV) capsid in place of the HIV-1 capsid to circumvent this restriction (J Virol. 2009). Transduction efficiency, however, remained highly variable between animals. In this study, we sought to evaluate the effects of rhesus TRIM5α polymorphisms on lentiviral transduction of rhesus hematopoietic repopulating cells.To evaluate whether rhesus TRIM5α polymorphisms influence the efficiency of lentiviral transduction, we transduced cell lines expressing 6 different rhesus TRIM5α types (Mamu-1, -2, -3, -4, -5, and TrimCyp) with eGFP-encoding HIV1, χHIV, and SIV vectors (Figure). Among all TRIM5α cell lines, transgene expression rates (%GFP) from the χHIV vector fell between that of the HIV-1 vector and that of the SIV vector. For the χHIV and SIV vectors, transduction efficiency was reduced in Mamu-1, -2, and -3 (p<0.01) expressing cell lines when compared to that of control cells. For the HIV-1 vector, there was a reduction in %GFP among all TRIM5α types (p<0.01). These results suggest that both the χHIV and SIV vectors escape restriction through rhesus TRIM5α Mamu-4, -5, and TrimCyp.We then analyzed 16 rhesus macaques who were transplanted with CD34+ cells transduced with the χHIV vector. We evaluated %GFP in granulocytes and lymphocytes 6 months after transplantation, as %GFP expression has been found to plateau in the peripheral blood at 6 months. Transduction efficiency of rhesus CD34+ cells was also evaluated in vitro at the time of transplant based on %GFP expression. For statistical analysis, we assessed factors that potentially affect transduction efficiency, including age, sex, weight, total number of mobilized CD34+ cells, cytokine mobilization regimen (G-CSF & stem cell factor (SCF) vs. G-CSF & plerixafor), cell density during transduction, and TRIM5α polymorphisms.Multivariable analysis demonstrated that TRIM5α type Mamu-4 (50.9±19.0% vs. 26.6±16.7%, p=0.04) as well as mobilization regimen (48.5±17.4% vs. 12.7±7.1%, p=0.01) affected CD34+ cell transduction efficiency in vitro. TRIM5α Mamu-4 only showed significant effects on %GFP among lymphocytes in vivo (23.7±17.9% vs. 5.3±3.1%, p=0.046). When analyzing the %GFP among granulocytes in vivo, there was a significant correlation with weight (p=0.01), mobilized CD34+ cell number (p=0.02), TRIM5α type Mamu-4 (29.3±25.4% vs. 8.4±6.0%, p=0.03), and mobilization regimen (25.4±24.1% vs. 7.5±3.0%, p=0.04). If in vitro %GFP is included in the analysis, both %GFP among granulocytes and lymphocytes are strongly affected (<0.001) and TRIM5α type Mamu-4 and mobilization regimen are no longer significant. Univariate analysis showed similar tendencies regarding %GFP among granulocytes and lymphocytes. Taken together, our data suggest that TRIM5α type Mamu-4, mobilization regimen (G-CSF & SCF), and CD34+ cell transduction efficiency in vitro are important factors that can predict higher %GFP in granulocytes and lymphocytes 6 months following transplantation.In summary, rhesus TRIM5α polymorphisms (especially type Mamu-4) play an important role in allowing efficient lentiviral transduction of long-term repopulating cells and contribute to the variable results observed in the rhesus hematopoietic stem cell gene therapy model. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Accelerated lymphocyte reconstitution and long-term recovery after transplantation of lentiviral-transduced rhesus CD34 + cells mobilized by G-CSF and plerixafor

Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor produces significant mobilization of CD34(+) cells in rhesus macaques. We sought to evaluate whether these CD34(+) cells can stably reconstitute blood cells with lentiviral gene marking. We performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34(+) cells transduced with a lentiviral vector, and these data were compared with those of G-CSF and stem cell factor mobilization. G-CSF and plerixafor mobilization resulted in CD34(+) cell yields that were twofold higher than yields with G-CSF and stem cell factor. CD123 (interleukin-3 receptor) expression was greater in G-CSF and plerixafor-mobilized CD34(+) cells when compared to G-CSF alone. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages, similar to animals who received G-CSF and stem cell factor-mobilized grafts. Lymphocyte engraftment was accelerated in animals receiving the G-CSF and plerixafor-mobilized CD34(+) cells. One animal in the G-CSF and plerixafor group developed cold agglutinin-associated skin rash during the first 3 months of rapid lymphocyte recovery. One year after transplantation, all animals had 2% to 10% transgene expression in all blood cell lineages. G-CSF and plerixafor-mobilized CD34(+) cells accelerate lymphocyte engraftment and contain hematopoietic stem cell capable of reconstituting multilineage blood cells. These findings indicate important differences to consider in plerixafor-based hematopoietic stem cell mobilization protocols in rhesus macaques.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

AbstractAbstract 1449Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Abstract 692HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press).To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation.These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Development of a Human Immunodeficiency Virus Type 1-Based Lentiviral Vector That Allows Efficient Transduction of both Human and Rhesus Blood Cells

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G, which target HIV-1 capsid and viral infectivity factor (Vif), respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV), including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs), the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs, the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus, 15 to 30% versus 1 to 5%; second rhesus, 7 to 15% versus 0.5 to 2%, respectively) 3 to 7 months postinfusion. In summary, we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application.

TransientIn Vivoβ-Globin Production After Lentiviral Gene Transfer to Hematopoietic Stem Cells in the Nonhuman Primate

Inherited disorders of globin synthesis remain desirable targets for hematopoietic stem cell (HSC)-based therapies. Gene transfer using retroviral vectors offers an alternative to allogeneic HSC transplantation by the permanent integration of potentially therapeutic genes into primary autologous HSCs. Although proof of principle has been demonstrated in humans, this approach has been met by formidable obstacles, and large-animal models have become increasingly important for the preclinical development of gene addition strategies. Here we report lentiviral gene transfer of the human beta-globin gene under the control of the globin promoter and large fragments of the globin locus control region (LCR) in the nonhuman primate. Using an HIV-1, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped vector, modified to overcome a species-specific restriction to HIV-1, gene transfer to colony-forming units (CFU) derived from mobilized peripheral blood (PB) rhesus CD34+ cells was 84.4 +/- 2.33%. Erythroid cells derived from transduced rhesus CD34+ cells expressed human beta-globin at high levels as assessed by flow cytometry with a human beta-globin-specific antibody. Two rhesus macaques (RQ3586 and RQ3583) were transplanted with mobilized PB CD34+ cells transduced with our modified HIV vector at a multiplicity of infection of 80. High gene transfer rates to CFUs were achieved in vitro (RQ3586, 87.5%; RQ3583, 83.3%), with efficient human beta-globin expression among erythroid progeny generated in vitro. Early posttransplantation, gene transfer rates of 5% or higher were detectable and confirmed by genomic Southern blotting, with equivalent-level human beta-globin expression detected by flow cytometry. Long-term gene marking levels among mononuclear cells and granulocytes assessed by quantitative polymerase chain reaction gradually decreased to about 0.001% at 2 years, likely due to additional HIV-1 restrictive elements in the rhesus macaque. No evidence of clonal hematopoiesis has occurred in our animals in up to 2 years. Current efforts are aimed at developing a lentiviral vector capable of efficiently transducing both human and rhesus HSCs to allow preclinical modeling of globin gene transfer.

Development of An HIV1-Based Lentiviral Vector Able to Transduce Both Human and Rhesus Blood Cells.

Although the successful application of gene transfer to hematopoietic stem cells using HIV based vectors for the hemoglobinopathies and thalassemias has recently been achieved in rodents, concerns regarding possible side effects, including insertional mutagenesis, mandate large animal preclinical testing. HIV vectors transduce rhesus blood cells poorly due to a species specific block by TRIM5a and APOBEC3G which target HIV capsid proteins (CAs) and viral infectivity factor (Vif), respectively. We developed a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV and SIV by including SIV CA and SIV Vif. Titers of HIV vectors prepared with SIV Vif (8.4±0.69x106 IU/ml, p=0.73) or SIV CA (delta-HIV 8.1±1.1x106 IU/ml, p=0.73) were comparable to that of a normal HIV vector (9.2±0.46x106 IU/ml), while titers of chimeric HIV vectors that included SIV gag-pol or rev-tat were dramatically decreased (0.18-4.5x105 IU/ml, p<0.001). Unlike the HIV chimeric vectors, chimeric SIV vectors that included both HIV gag-pol and rev-tat showed relatively high titers when compared to normal SIV vectors (p=0.88), while titers of chimeric SIV vectors that included HIV CA were very low (p<0.01). To evaluate whether the candidate chimeric HIV vectors were able to transduce both human and rhesus blood cell lines, we performed dose-escalation transductions using the chimeric vectors. The delta-HIV vector showed superior transduction of human blood cell lines (61±2.8% at MOI=3, p<0.01) than the SIV vector (49±1.8%) and could efficiently transduce rhesus blood cell lines (18±0.90% at MOI=7, p<0.001) while the HIV vector could not (1.8±0.05%). To evaluate the transduction efficiency of delta-HIV on human and rhesus hematopoietic cells, we transduced human and rhesus CD34+ cells with delta-HIV and HIV vectors at various MOIs (0.5–50). In human CD34+ cells, the delta-HIV and HIV vectors showed similar transduction efficiency at lower MOIs (51±1.3%, 47±1.7% at MOI=5, p=0.18, respectively), while in rhesus cells, the transduction rates of delta-HIV (48±1.4% at MOI=2) were significantly superior to that of the HIV vector (6.5±0.36% at all MOIs, p<0.01). Among rhesus CFU, the delta-HIV vector produced a significantly higher percentage of GFP positive clones in comparison to those transduced with the HIV vector (Erythroid (E): 77±1.0% vs. 15±2.7% p<0.01, Myeloid (M): 46±1.8% vs. 1.2±0.59% p<0.01, respectively). On the other hand, among human CFU, similar rates of transgene expressing clones were observed (E: 79±0.79% vs. 72±0.64% p<0.01, M: 34±2.0% vs. 37±0.88% p=0.22, respectively). In erythroid and myeloid liquid cultures derived from transduced CD34+ cells, rhesus cells showed higher rates of transgene expression after delta-HIV transduction than after HIV transduction (E: 51±6.4% vs. 11±0.24% p<0.001, M: 27±2.5% vs. 6.1±2.0% p<0.001, respectively). In human erythroid and myeloid cells, the transgene expression rates were similar (E: 66±4.0% vs. 68±2.3% p=0.61, M: 36±1.5% vs. 37±1.5% p=0.93, respectively). In summary, we have developed an HIV-based lentiviral vector system capable of efficient transduction of both human and rhesus blood cells. This vector system should allow preclinical testing of HIV based therapeutic vectors in the large animal model. [Display omitted]

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Initial evaluation of oncoretroviral vectors carrying HIV-1 inhibitor gene into rhesus CD34+ cells and/or CD4+ T cells: An in vivo model for the gene therapy of AIDS

Initial evaluation of oncoretroviral vectors carrying HIV-1 inhibitor gene into rhesus CD34+ cells and/or CD4+ T cells: An in vivo model for the gene therapy of AIDS

Distinctive Integration Profile of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-Term Repopulating Cells.

Insertional mutagenesis continues to be a major concern in hematopoietic stem cell gene therapy. Non-conventional gene transfer vectors with more favorable integration features in comparison to conventional retrovirus and lentivirus vectors are being pursued. The avian sarcoma leukosis virus (ASLV) has been reported to have an unbiased integration pattern in cell lines, but this vector system has not previously been investigated for transduction of repopulating hematopoietic cells. We investigated the efficiency and integration profile of this vector in the clinically-relevant rhesus macaque autologous transplantation model. Using an ASLV derived RCAS (Replication Competent, ALV LTR with A Splice acceptor) vector, we could obtain transduction efficiencies of up to 33% ex vivo in rhesus CD34+ cells. We have been able to transplant two rhesus macaques with ASLV-transduced autologous CD34+ cells and achieve long-term gene marking levels of 1–3% as far as 18 months post-transplant. Using an optimized linear-amplification mediated PCR (LAM-PCR), we have been able to identify so far close to 300 unique insertion sites in the two animals. Here we reported for the first time a systematic analysis of 239 ASLV insertion sites identified in rhesus long-term repopulating cells. Out of 239 unique insertions identified in 4–18 months post-transplant granulocytes and lymphocytes, 99 (41.1%) have landed within RefSeq gene coding regions. 14 (5.8%) have landed within 5kb upstream of RefSeq genes, indicating no preference of inserting into transcription start site. 10 (4.2%) have landed within 5kb downstream of RefSeq genes, which is comparable to random insertions. No insertion into the Mds1-Evi1 locus has been identified to date, at any time point, which is in striking contrast to significant overrepresentation of this insertion site for MLV vectors in the same transplantation model. No preference into CpG islands was found. We further employed a rapid and quantitative assay for measuring neighboring gene activation by vector provirus LTR enhancers using a luciferase assay. LTR from vectors are cloned into the pACT5 plasmid, upstream of a minimal promoter-IRES-luciferease cassette, allowing measurement of neighboring gene activation by a simple luciferase assay. RCAS vectors produced no detectable luciferase activation, both in the forward and reverse orientations, suggesting lack of enhancer activity in the LTRs in mammalian cells, in contrast to significant enhancer activity and read-through transcription from MLV vectors measured in the same assay. Our findings indicate that ASLV derived RCAS vectors have a potentially safer integration pattern in comparison to commonly used MLV and HIV derived vectors. Furthermore, ASLV LTRs do not have significant promoter and enhancer activity in mammalian cells, which provide a second safety feature of the system. Therefore, these vectors should be further explored for hematopoietic stem cell gene transfer purposes.

Potent Inhibition of Simian Immunodeficiency Virus (SIV) Replication by an SIV-Based Lentiviral Vector Expressing Antisense Env

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

Long-Term Polyclonal and Stable Gene Transfer into Rhesus Repopulating Hematopoietic Stem Cells Using a Simian Immunodeficiency Virus-Based Lentiviral Vector System.

Murine leukemia virus (MLV) vectors have been studied extensively in animal models and utilized for over a decade in clinical trials of gene therapy directed at hematopoietic stem and progenitor cells MLV have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression. More recently concerns have arisen regarding their safety regarding activation of adjacent proto-oncogenes and resultant leukemogenesis. We have previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus CD34+ cells, resulting in high-level in vivo marking with transduced progeny cells up to one year post-transplantation.(Hanawa et al, 2004) A comparison of vector integration sites in these animals compared to animals receiving MLV-transduced cells revealed different patterns, showing that SIV integrants strongly favored entire transcription units and gene-dense regions of the genome, compared to MLV that favored regions surrounding transcription start sites.(Hematti et al, 2004). Animals receiving MLV-transduced cells had highly non-random engraftment with integrants in or near the the MDS1/EVI1 gene complex. To evaluate long-term safety implications of the SIV vector-mediated CD34+ cell gene transfer, we analyzed the insertional sites in granulocytes, T cell, and B cells from 3 rhesus macaques which were transplanted three years ago with transduced, autologous cytokine-mobilized peripheral blood CD34+ cells. All three animals continued to show significant marking and expression levels in T cells, B cells and granulocytes, with mean GFP + levels of 6.7% (range, 3.3–13.0%), 7.4% (4.2–13.4%) and 5.6% (3.1–10.5%), respectively. Vector insertion site analysis by linear amplification-mediated PCR method at three years continued to show highly polyclonal reconstitution. Subsequent cloning and sequencing data confirmed long-term polyclonality with vector-containing cells and there was no evidence for any worrisome common integration sites, with no integrants detected in the MDS1/EVI1 region, in contrast to results with the MLV vector. These results indicate that the SIV vector system can result in stable and efficient long-term expression in progeny of transduced CD34+ cells, without the worrisome integration profile previously reported in our model with MLV vectors.

368. Lentiviral Vector Strain of Origin and Target Cell Species-Specific Restriction Factors Influence Transduction Efficiency

The efficiency of transduction of both human and non-human primate cells by lentiviral vectors is influenced by interactions with members of the TRIM5|[alpha]| family of cytoplasmic proteins. This has been a particular problem when using HIV-1-derived vectors in rhesus macaque hematopoietic cells, where the vectors are strongly restricted by rhesus TRIM5|[alpha]|, although SIV-derived vectors are not. Current models to explain this variation in restriction postulate that the recruitment of cyclophilin A (CypA) by HIV-1 capsid protects the virus from TRIM5|[alpha]| restriction in human cells, but not in macaque cells. Conversely, although the SIVmac capsid does not bind CypA, it is not significantly restricted by human TRIM5|[alpha]|. We wanted to further investigate the role of the CypA-capsid interaction in the restriction of lentiviral vectors by transducing various cell types with VSV-G-pseudotyped lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) gene and then analyzing gene expression by FACS. As expected, we found that treatment of human embryonic kidney cells (HEK293A) cells with cyclosporine A (CsA), which inhibits the CypA-capsid interaction, resulted in a |[sim]|2-fold drop in transduction with the HIV-1-based vector. Inclusion of either the HIV-2 or SIV nef proteins in the vector did not relieve its sensitivity to CsA. In contrast, transduction with HIV-1 vectors containing certain capsid substitutions within the CypA-binding site (Chatterji et al., JBC;280(48):40293|[ndash]|40300) was not negatively affected by CsA treatment, suggesting that the mutated vectors no longer require CypA for efficient transduction of human cells. These vectors also transduced owl monkey and rhesus macaque kidney cells more efficiently than the wild-type vector. Vectors derived from different SIVmac-strains were found to transduce rhesus macaque cell lines at equal efficiency, but transduction of primary rhesus CD34+ cells with an SIVmac1A11-derived vector (Hanawa et al., Blood;103:4062|[ndash]|4069) was significantly more efficient (30|[ndash]|60 %) than with an SIVmac251-derived vector (4|[ndash]|12%) (N|[egrave]|gre et al., Gene Therapy;7:1613|[ndash]|1623). Experiments in which the SIVmac transfer vectors were crosspackaged with capsids from the other SIVmac strains revealed that this property maps to the transfer vector and not to the packaging construct. CsA treatment increased transduction with SIVmac-derived vectors in both rhesus and owl monkey cell lines and in primary human CD34+ cells, suggesting that the SIVmac capsid is susceptible to restriction in these tissues. In contrast, CsA treatment had no effect on transduction with either HIV-1 or SIVmac-derived vectors in primary rhesus macaque CD34+ cells. Together, these results show differential restriction of lentiviral vectors that varies with the species and cell type transduced. These findings may be relevant for studies using lentiviral gene transfer into non-human primate models.

Inhibition of Simian/Human Immunodeficiency Virus Replication in CD4+ T Cells Derived from Lentiviral-Transduced CD34+ Hematopoietic Cells

We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34+ bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34+ cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4+ GFP+ T cells derived from VRX494-transduced CD34+ cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.

1030. A Rhesus Macaque Transplant Model for Drug Selection of Hematopoietic Stem Cells Transduced with MGMT Vectors

Gene transfer of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) into hematopoietic stem cells (HSCs) results in efficient in vivo selection of murine HSCs after treatment with temozolomide (TMZ) and O6-benzylguanine (BG). In addition, MGMT gene transfer into HSCs can protect hematopoiesis from the toxic side effects of chemotherapeutic agents in the treatment of malignant diseases. We are studying whether this drug resistance system will be efficacious in non-human primates using a rhesus macaque autologous transplantation model. In our first two transplant cases, we used an MSCV-P140K-IRES-GFP vector to transduce bone marrow cells that had been harvested after treatment with SCF and G-CSF, and then purified on a Miltenyi CliniMACS column using the anti-CD34 MoAb clone 563. Both animals engrafted after total body irradiation. The first animal showed relatively high levels of GFP+ B cells (40%) and lower levels of GFP+ granulocytes (3%) in the peripheral blood prior to treatment with 2 courses of TMZ and BG, 88 and 98 days after transplant. After the second course of drug selection, there was an increase in GFP+ granulocytes from 3% to a peak of 18%. These levels gradually declined back to baseline over the next 75 days. Subsequent treatment courses did not result in further selection. The second transplant case had less than 1% GFP+ granulocytes in the peripheral blood, probably due to an insufficient dose of total body irradiation, and no evidence of in vivo selection following drug treatment. In order to achieve higher levels of marking prior to selection, and to test whether inclusion of HOXB4 would improve the efficiency of selection, we developed a series of SIV vectors which co-expressed various combinations of P140K-MGMT, HOXB4, and GFP using the self-cleavage peptide 2A from porcine teschovirus-1 (P2A). A tricistronic SIV vector was constructed that incorporated all three genes by using different 2A sequences from the porcine teschovirus-1 and the asigna virus. Western blot analysis showed complete cleavage of each protein with no fusion proteins detected. We then generated amphotropic pseudotyped vectors with average titers ranging from 2 |[acute]| 105 to 1.2 |[acute]| 106 per milliliters on HeLa cells. Transduction of rhesus CD34+ cells with these SIV vectors resulted in around 40% GFP+ cells immediately after transduction for the MGMT-P2A-GFP vector and the MGMT-P2A-HOXB4-T2A-GFP vector, and about 20% marking for the HOXB4-P2A-GFP vector. In the presence of 800 mM TMZ, about 25% of CFU-C were drug resistant in vitro using the SIV vectors containing the MGMT expression cassette. For both vectors expressing HOXB4, expansion of GFP+ CD34+ cells was noted over 12-day in vitro cultures, with GFP+ cells increasing from 20% to 40% in most experiments. Our efforts are now focused on further evaluation of these SIV vectors in vivo, in transplanted rhesus macaques.

62. Transduction of CD34+ Hematopoietic Progenitor Cells with a SIV-Based Lentiviral Vector Expressing Antisense SIV Env Protects CD4+ T Cell Progeny from SIVmac239 Infection

In light of recent findings demonstrating that the rhesus TRIM-5a protein specifically binds to HIV-1 Gag and blocks the uncoating of viral particles, SIV-based lentiviral vectors have been proposed as a more appropriate vector than HIV-1 based vectors for the transduction of rhesus macaque hematopoietic stem cells. We evaluated the ability of a SIVmac239-based lentiviral vector (VRX859), which encodes 1275 bp antisense SIV envelope and eGFP transcriptionally regulated from the SIV LTR, to inhibit viral replication in vitro and compared transduction of rhesus CD34+ lymphoid progenitor cells with an HIV-1 based lentiviral vector. Initially, we evaluated inhibition by VRX859 of the homologous SIVmac239; the related SIVmac251; the heterologous SIVsmE660 (|[sim]|80% homologous in env); the chimeric SHIV 89.6P and HIV-1 NL4-3. CEMx174 cells, which support replication of both HIV-1 and SIV isolates, were transduced with VRX859 at MOI of 10 and the eGFP sorted population contained 1 proviral copy per cell. Following infection with SIVmac239 and SIVmac251, CEMx174-VRX859 cells exhibited over 1000- and 650-fold inhibition of viral replication compared to control cells. Inhibition of SIVsmE660 and SHIV 89.6P was weaker, ranging from 3 to 9-fold and 18 to 32-fold. HIV-1 NL4-3 replication in CEMx174-VRX859 cells was similar to control cells. To evaluate transduction of rhesus CD34+ cells and lymphoid progenitor cells, we prestimulated CD34+ cells with Tpo, Flt3L and SCF (50 ng/ml each) for 1 day and exposed cells to VRX859 vector (or the analogous HIV-1 based lentiviral vector VRX494) for 1, 2 or 3 days on Retronectin-coated plates. Transduction of rhesus CD34+ cells with the SIV vector VRX859 over a range of MOIs (3|[ndash]|50 IU per cell) resulted in similar percentages of eGFP expression as observed with the HIV vector VRX494 at similar MOIs. Transduced CD34+ cells were then cultured with rhesus fetal thymus stromal cells to induce T cell differentiation and analyzed by flow for expression of eGFP. eGFP expression in CD4+ T cells derived from CD34+ cells transduced with VRX859 at low MOIs (3|[ndash]|10 IU per cell) was increased from 13- to 2.2- fold compared cells transduced with VRX494. eGFP expression in CD4+ T cells derived from CD34+ cells transduced at higher MOIs (30 and 50 IU per cell) were basically equivalent between VRX859 and VRX494. Rhesus CD4+ T cells expressing eGFP were obtained by cell sorting and expanded with ConA, IL-2 and irradiated human feeders. GFP+ CD4+ T cells derived from VRX859-transduced cells strongly inhibited SIVmac239, SIVsmE660 and SHIV 89.6P replication (up to 1000-fold) as compared to control CD4+ T cells. These results demonstrate superior transduction of rhesus lymphoid progenitor cells with a SIV-based lentiviral vector, particularly at low MOI, and strong inhibition of viral replication, based partially on competition with genomic RNA, in CD4+ T cells derived from transduced CD34+ cells and in a CD4+ cell line. These results support using a SIV-based lentiviral vector in the rhesus macaque model as a valuable preclinical model for stem cell gene therapy for AIDS.

Lentiviral Vector-Transduced Dendritic Cells Induce Specific T Cell Response in a Nonhuman Primate Model

Dendritic cells (DCs) are effective in stimulating and controlling the outcome of T cell responses. Human immunodeficiency virus type 1-based lentiviral vectors can achieve sustained transduction of genes/antigens in dividing and nondividing cells, thus representing a candidate vector for stable expression of antigens in DCs. We previously established conditions for transduction of purified cytokine mobilized rhesus CD34(+) cells in vitro, and transplantation of the autologous transduced cells in a nonhuman primate model in vivo. In the present study, we transplanted DCs derived from EGFP-transduced CD34(+) cells into nonmyeloablated rhesus macaques. Transplantation of DCs stably expressing EGFP into autologous animals induces persistent, long-lived (up to 100 weeks) EGFP-specific T cell responses. Of note, no humoral responses against EGFP are detected in the transplanted animals. These studies provide, to our knowledge, the first demonstration that lentiviral transduction of CD34(+) progenitor cells subsequently differentiated to DCs is capable of priming a specific T cell response in a nonhuman primate in vivo. Taken together, our data provide formal in vivo evidence that lentivirus-transduced dendritic cells represent a potential approach in eliciting cellular immune responses in primates.

T-cell differentiation of human and non-human primate CD34+ hematopoietic progenitor cells using porcine thymic stroma.

Transplantation of swine thymic tissue has been proposed as an approach to reconstitute the immune system of HIV-infected individuals. This is an attractive strategy because miniature swine are readily available as donors and porcine tissue is resistant to infection with HIV-1. Demonstration that porcine thymus tissue supports primate T-cell differentiation is critical to the ultimate utility of this approach. Using a thymic stroma culture system we have previously described [Rosenzweig M, Marks DF, Zhu H et al. In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells. Blood 1996; 87: 4040], we demonstrate that porcine thymus tissue is able to promote the in vitro T-lymphocyte differentiation of both human and non-human primate hematopoietic progenitor cells. CD34+ hematopoietic progenitors differentiated into both double positive (CD4+CD8+) and single positive thymocytes expressing CD4 or CD8 alone. A polyclonal T-cell repertoire was evident. In addition, the T cells responded appropriately to mitogen and were permissive to infection with simian immunodeficiency virus (SIV). These data demonstrate the ability of porcine thymus to support T-cell differentiation of both human and non-human hematopoietic progenitor cells and support in vivo studies of transplantation of swine thymic tissue as a strategy for immune reconstitution in AIDS.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.